The present invention relates to a dynamic continuous flow enzyme reactor and method which allow in vitro measurement of the rates of blood coagulation reactions and other dynamic enzymatic reactions which are dependent for activity on phospholipids in an environment closely approximating that found in the body.
The coagulation (clotting) system in man and animals is a major contributor to the maintenance of hemostasis and also to thrombus (blood clot) formation. Coagulation is essentially a cascade in which each clotting factor, which is normally present in the blood and other tissues as an inactive enzyme precursor, i.e., zymogen, is in sequence activated into a proteolytic enzyme that selectively attacks the next zymogen in the sequence, thereby converting it into an active enzyme. Amplification occurs at each step in the process so that a small initial stimulus can ultimately result in a significant amount of fibrin clot.
The clotting cascade begins as two separate pathways that ultimately converge. One pathway is "intrinsic" to the blood and the other one is termed "extrinsic" because it is triggered by clotting factors not normally present in blood. The intrinsic pathway plays a major role in hemostasis following injury. The extrinsic pathway can become activated in a variety of pathologic situations, e.g., diffuse endothelial damage, advanced cancer, endotoxemia, and pregnancy complications.
There is now considerable evidence that coagulation is started in the body when factor VII, a vitamin K-dependent plasma clotting factor protein and tissue factor, a cellbound protein not normally associated with blood cells, interact. (See e.g. Nemerson, Blood 71:1-8, 1988 for a review). This interaction results in an activated complex which has enzymatic activity and initiates clotting by converting two other proteins, i.e., factor X and factor IX, to their active, enzymatic forms, factor X.sub.a and factor IX.sub.a, respectively. (In accord with common practice, precursor, i.e., the zymogen, forms of the active blood clotting factors are denoted by a Roman numeral, and the active forms are indicated by a subscripted "a", e.g. factor X for zymogen and factor X.sub.a for activated factor.)
Tissue factor is a procoagulant protein present on the surface of virtually all cells, not normally in direct contact with blood. However, tissue factor is inducible in endothelial cells and monocytes upon stimulation with various pharmacologic mediators, e.g. tumor necrosis factor, interleukin-1 and endotoxin. The extrinsic coagulation pathway is triggered by tissue factor which complexes with and activates factor VII, a vitamin K-dependent serine protease zymogen. The activation of factor VII by tissue factor occurs in the presence of calcium ions and is believed to result from a conformational change in factor VII. See, e.g., Nemerson et al. (1982) in Progress in Hemostasis and Thrombosis, Spaet, T. H. edit., Grune & Stratton, New York, vol. 6, pp. 237-261; Carson (1984) Prog. Clin. Pathol. 9:1-14. Conversion of the factor VII zymogen to the factor VII.sub.a active enzyme is accomplished by cleavage of an arg-ile peptide bond in the zymogen resulting in factor VII.sub.a which has a light chain containing the Gla region and a heavy chain that contains the enzyme active site.
If the zymogen factor VII had procoagulant activity, then the initiation of coagulation could simply follow upon the breaking of a physical barrier that normally separates factor VII from tissue factor. Thus, for hemostatis to occur, the injury itself may be sufficient to initiate coagulation. The determination that a zymogen has a small amount of activity relative to its derivative enzyme is fraught with difficulty because an active zymogen would have the same activity as an inert zymogen contaminated with a trace amount of an enzyme. In most instances, this problem is approached simply by treating the zymogen with an active site-directed enzyme inhibitor such as diisopropylfluorophosphate (DFP) or an appropriate chloromethylketone, thereby inhibiting the contaminating enzyme. Because zymogens usually are almost inert, this results in a total loss of measurable activity. However, the factor VII zymogen is itself readily inhibited by DFP, thus obviating this straightforward approach. Indeed, the reactivity of factor VII toward DFP is so great that by itself, it suggests extraordinary activity of the factor VII zymogen.
The DFP inhibition studies using bovine factors VII and VII.sub.a, showed that factor VII qualitatively has the same enzymatic activity as factor VII.sub.a although the factor VII zymogen contains slightly less than 1% of the activity of factor VII.sub.a. DFP has also been shown to inhibit human factor VII, the rate being one third of that for the inhibition of factor VIIa, which is about the same ratio observed when bovine proteins were used. See, e.g. Nemerson, Blood 71:1-8, 1988 and Zur et al., J. Biol. Chem. 257:5623-5631, 1982.
Experiments support the notion that coagulation can be initiated simply by the physical complexation of tissue factor and factor VII. Further evidence for this concept is derived from the observation that bovine factors VII and VII.sub.a bind to tissue factor with essentially the same dissociation constants. When monocytes were used as a source of human tissue factor, the same phenomenon was observed for human factors VII and VII.sub.a. Accordingly, one need not postulate a proteolytic initiation of coagulation, thereby avoiding the problem of an infinite regression of proteolytic events. This degree of activity of the zymogen is unusual in general and appears to be unique in the clotting system. The activity of factor VII or, indeed factor VII.sub.a is compatible with a quiescent coagulation system because in the absence of tissue factor it cannot trigger coagulation.
Owing to its intrinsic reactivity, factor VII is distinguished from all other known clotting zymogens. Thus, because the factor VII zymogen has enzymatic activity, when both the zymogen and active enzyme are referred to without distinguishing between the two species, the designation of factor VII(a) is used.
Tissue factor is likewise unique among the cofactors because, in contrast to the clotting factors V and VIII and other cofactors in the clotting cascade, the mature tissue factor protein apparently requires no further processing for its activity. These observations taken together suggest that the only requirement for the initiation of coagulation by tissue factor is its physical complexation with factor VII.
Tissue factor, which is a membrane-bound glycoprotein associated with phospholipids, is not normally present in the circulation. When blood vessels are disrupted, however, factor VII, which is a plasma coagulation factor, can complex with tissue factor, thereby forming a catalytically-active species which activates both factor IX (plasma thromboplastin component) a component of the intrinsic pathway to form factor IX.sub.a and factor X (Stuart factor), which is involved in both the extrinsic and intrinsic pathways of coagulation, to yield factor X.sub.a. Tissue factor also has important clinical use as a diagnostic reagent to monitor and study clotting.
Factor VII is present in trace amounts in the plasma (ca. 10 nM). The severe bleeding seen in individuals who are markedly deficient in factor VII demonstrates the physiologic importance of this protein. Deficiencies of factor VII are rare, but recent evidence suggests that some 16% of affected patients have cerebral hemorrhages usually resulting in death. Ragni et al., Factor VII Deficiency, Amer. J. Hematol., 10:79-88 (1981). On the other hand, patients with as little as 5% of the normal levels of factor VII sometimes have little or no hemorrhagic symptoms. For any given factor VII level, however, there is considerable clinical variability.
A variety of disorders, e.g. cancer and cardiovascular disease, are associated with increases in blood clot formation in the blood vessels. A main treatment for cardiovascular disease involves the use of anticoagulants, e.g. warfarin and related drugs, which interfere with the synthesis of vitamin K-dependent clotting factors (e.g., factors II, VII, IX and X). There are many studies which indicate that this treatment decreases the incidence of venous thromboembolism, pulmonary embolism and myocardial infarction (heart attacks). However, warfarin therapy is also associated with a rather high incidence of hemorrhage, which is sometimes fatal.
The standard way in which the dosage of the warfarin-type anticoagulants is monitored is by using the Quick one-stage prothrombin time. In this test, which is performed under static conditions, a sample of the patients blood plasma is warmed to 37.degree. C. A suspension of tissue thromboplastin (crude tissue factor) is then added to the plasma sample together with calcium ions and the clotting time is determined. Normal clotting time is 12+/-0.5 seconds. The therapeutic range of the anticoagulant is a blood concentration of the drug which provides a clotting time which ranges between 1.2 and 1.5 times the normal value. This narrow range imposes on clinical laboratories a precision which is frequently not attainable. These inaccuracies are believed to be responsible for some of the hemorrhagic side effects of the anticoagulant drugs.
Factor VII can be measured in a similar manner. Dilutions of the patient's plasma are added to normal plasma and the one-stage prothrombin time test is performed. The amount of factor VII present in the test sample is estimated by comparing the clotting times of the test samples with those obtained from dilutions of normal plasma. Indeed, to date all tests of the coagulation system have been based on the determination of clotting times of various plasmas, but always in a test tube under static conditions. However, because blood coagulation in vivo always occurs in a moving stream, the effects of flow on the enzymatic reactions cannot be properly evaluated in a static system.
It has now been found that the specific blood clotting enzymatic reactions can be more specifically performed in a dynamic fashion by passing various blood clotting zymogens together with calcium ions at a defined flow rate through a tubular housing member which is coated on its inner surface with a planar phospholipid bilayer membrane. Optionally and preferably the planar membrane has purified tissue factor incorporated therein. The reagents passed through the housing include either factor VII or factor VII.sub.a, which complexes with the tissue factor to form an enzymatically active species, together with factor IX or factor X which are the substrates for the tissue factor--factor VII complex. The rates of factor IX.sub.a or factor X.sub.a production can be readily analyzed by any suitable assay for these factors. The dynamic reaction allows for more specific analysis of the production of activated factors than can be obtained by current static methods.
Furthermore, passage of a plasma sample, e.g. from a patient, through such a phospholipid membrane-coated device under defined flow rates and other conditions of the invention allows the measurement of specific enzymatic products produced by interaction of the plasma sample with the tissue factor-containing phospholipid membrane and can provide valuable information on deficiencies of specific clotting factors or a more sensitive monitoring of proper anticoagulation parameters in patients than was obtainable by prior methods.
The enzyme reactor of the invention may also be used for carrying out and analyzing other phospholipid-dependent enzymatic reactions other than blood clotting reactions. Such reactions involve flowing various inactive enzyme reaction components in the reagent solution through a phospholipid bilayer membrane-coated tubular housing. The inactive enzyme components in the reagent solution become enzymatically active by interaction with the phospholipid membrane on the inner surface of the housing and the products of the reaction can be analyzed in the effluent solution.